Administration of a thiol-based chemoprotectant compound

ABSTRACT

A method of administration of a thiol-based chemoprotectant agent including NAC (N-acetylcysteine) and STS (sodium thiosulfate) that markedly affects biodistribution and protects against injury from diagnostic or therapeutic intra-arterial procedures. A method for treating or mitigating the side effects of cytotoxic cancer therapy for tumors located in the head or neck and brain tumors. The thiol-based chemoprotectant agent is administered intra-arterially with rapid and first pass uptake in organs and tissues other than the liver.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The present invention was partially supported under NIH grant No.NS33618 and by The Department of Veterans Affairs merit review grant.The United States Government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

The present invention is based, in part, upon the discovery that amethod of administration of N-acetylcysteine (NAC) markedly affects itseffective biodistribution. The present invention provides a method fortreating or mitigating the side effects, including organ damage, ofcytotoxic cancer therapy for tumors located in the head or neck.Additionally, NAC or other thiols can be administered concurrently with,before or after, intra-arterial procedures and provides protectiveaffects to prevent or diminish organ damage.

N-acetylcysteine (NAC) is an analog of cysteine. When NAC isadministered to a mammal it is deacylated and enters a cellularsynthetic pathway for the production of glutathione. Glutathione isinvolved in the cellular pathways influencing a tumor's resistivity tocytotoxic drugs. The cytotoxic properties of chemotherapeutic drugs canbe enhanced by pretreatment with buthionine sulfoximine (BSO) therebyreducing intracellular glutathione. However, reduction of intracellulargluthionine will potentiate systemic toxicities associated withchemotherapeutic drugs. Thus, this procedure is dose-limiting. Forprotection, the glutathione levels of “normal” cells have to bereestablished if BSO is used to potentiate the cytotoxic properties ofcytotoxic cancer therapies (Kamer et al., Cancer Res. 47:1593-1597,1987; Ozols et al., Biochem. Pharm. 36:147-153, 1987; McLellan et al.,Carcinogenesis 17:2099-2106, 1995; and Shattuck et al., J. ParenteralEnteral Nutrition 24:228-233, 1998). It may be possible to reduce thebone marrow toxicity of chemotherapeutic drugs by usingsulfur-containing chemoprotective agents (thio, thiol, and thioethercompounds) to mimic one or many of the activities of glutathione such asconjugation, free radical scavenging, and drug efflux via the multidrugresistance associated proteins. NAC and other thiol agents such as STShave early detoxifying activity not related to the later increase inglutathione levels. These early detoxifying effects occur because thethiols themselves mimic some actions of glutathione such as free radicalscavenging, anti-oxidant activity, chemical conjugation, and activationof efflux pumps.

A potential problem with any chemoprotectant is the possibility ofdeactivating the anti-tumor effect of the chemotherapy or radiationtherapy. The goal of chemoprotection is to reduce unwanted toxicities ofchemotherapy or radiotherapy without affecting efficacy.

For brain tumor chemotherapy, one must attempt to increase the deliveryof chemotherapy to the brain tumor and block the delivery of thechemoprotective agent. Additionally, one will want to target thechemoprotectant agent to the bone marrow to protect againstmyelosuppression and to liver, kidney and lung to prevent organtoxicity. Therefore, there is a need in the art to improvepharmacokinetics and biodistribution of chemoprotectant agents so thatthey will be more effective when delivered in a tissue-specific manner.Preferably, delivery is maximized to the bone marrow, chest and abdomenorgans while minimized to the brain.

There are more than 10 specific active transport systems that transportcompounds from the blood to the brain. Otherwise, substances, such aschemoprotectants can only nominally penetrate this barrier by passivediffusion. Brain tumors are particularly difficult to treat because theblood-brain barrier is an anatomical structure that limits the egress ofconstituents in the blood to the brain. Thus, brain tumors often respondpoorly to chemotherapeutic drugs. There have been many attempts to tryto increase brain bioavailability of various drug compounds to braintissue. One technique uses osmotic BBB modification by administeringmannitol through the internal carotid artery (Neuwelt et al., CancerRes. 45:2827-2833, 1985). This technique is useful for administering thechemotherapeutic methotrexate to experimental brain tumors that wouldotherwise be inaccessible to this drug (it poorly crosses the BBB). Theosmotic shrinkage caused by intracarotid mannitol administration allowedfor temporary BBB disruption and increased tumor delivery of themethotrexate. Thus, a temporary disruption of the barrier functions ofthe BBB can be induced by a sugar, such as mannitol, and cause higherbrain concentrations of a drug compound that would not otherwise havecrossed the BBB. This BBB opening technique has also been investigatedwith other chemotherapeutic drugs (Neuwelt et al., Proc. Natl. Acad.Sci. USA 79:4420-4423, 1982; Fortin D. McCormich Cl, Remsen L G, NixonR, Neuwelt EA, “Unexpected neurotoxicity of etoposide phosphate whengiven in combination with other chemotherapeutic agents afterblood-brain barrier modification using propofol for general anesthesiain a rat model,” Neurosurgery 47:199-207, 2000).

An example of chemoprotection is a drug neutralization techniquedescribed in U.S. Pat. No. 5,124,146 wherein excess toxic drug compoundsare “mopped up” or bound by a binding or neutralizing agent not able topenetrate the blood brain barrier. This technique requires precisetiming as to when the drug neutralizing agent is administered.

There are several thiol-based chemoprotectant agents that contain athio, thiol, aminothiol or thioester moiety. Several thiol-basedchemoprotectant agents have been shown to provide protection against atleast some of the systemic toxicities caused by alkylatingchemotherapeutics. The thiol based chemoprotective agents includeN-acetyl cysteine (NAC), sodium thiosulfate (STS), GSH ethyl ester,D-methionine, and thiol amifostine (Ethyol or WR2721). NAC is currentlymarketed in the United States under an orphan indication for oral andintravenous (i.v.) administration for overdosing with acetaminophen. NAChas also been shown to be a chemoprotectant when administered incombination with a vanadate compound (U.S. Pat. No. 5,843,481; and Yarbo(ed) Semin. Oncol. 10 [Suppl 1]56-61, 1983). Ethyol is also marketed inthe United States under the generic name of Amifostine. GSH ethyl esteris an experimental thiol not yet marketed for clinical use, but isrepresentative of the class of thiols that is converted directly toglutathione.

In addition, NAC has been shown to be a mucoregulatory drug used for thetreatment of chronic bronchitis (Grassi and Morandini, Eur. J. Clin.Pharmacol. 9:393-396, 1976; Multicenter Study Group, Eur. J. Respir.Dis. 61: [Suppl.] 93-108, 1980; and Borman et al., Eur. J. Respir. Dis.64:405-415, 1983).

In plasma, NAC can be present in its intact, reduced forms as well as invarious oxidized forms. It can be oxidized to a disulfide by reactingwith other low molecular weight thiols, such as cysteine andglutathione. NAC can be oxidized by reacting the thiol groups of plasmaproteins. When administered intravenously, the brain levels of NAC are<5%. Yet, NAC does cross the BBB if given by an intra-arterial route ofadministration. NAC is rapidly cleared from plasma via the liver andkidney. Moreover, NAC does not show neurotoxic properties.

There are bioanalytical methods for the determination of NAC in plasma,including Cotgreave and Moldeus, Biopharm. Drug Disp. 8:365-375, 1987;and Johansson and Westerlund, J. Chromatogr. 385:343-356, 1986 that alsopermit a determination of other forms of NAC. Moreover, cysteine andcystine have been identified as major metabolites of NAC. The excretedurinary product is inorganic sulfate together with small amounts oftaurine and unchanged NAC. According to the label indications for NACmanufactured by (American Regent Laboratories Shirley, N.Y.), vials ofNAC are produced as a sterile solution for oral administration dilutedwith water or soft drinks.

Another thiol-containing chemoprotectant is sodium thiosulfate (STS).Its chemical formula is Na₂S₂O₃ and it has been used clinically forcyanide poisoning and for nephrotoxicitiy caused by cisplatin. STS iscleared rapidly from circulation primarily by the kidney. The plasmahalf life after a bolus injection is about 17 minutes. STS can alsoinactivate platinum agents through covalent binding to platinum agentsat a molar excess >40:1 (STS:platinum). With i.v. administration of STS,the brain levels of STS are <5% of blood showing poor brainlocalization. Neurotoxic side effects, in the form of seizures, mayoccur when brain levels of STS are enhanced through i.a. administrationwithin 30 min of BBB disruption.

Diagnostic or therapeutic procedures involving intra-arterialcatheterization can cause a variety of organ toxicities, complicationsand side effects from injuries. For example, placement of an arterialcatheter can dislodge plaques from artery walls that can lodge elsewherein the vasculature causing ischemia. Ischemia increases the presence offree radicals and leads to cell death. As another example,nephrotoxicity of radiographic contrast agents can lead to acute renalfailure even when measures are taken to reduce toxic effects. As a thirdexample, intra-arterial catheterization is used during angioplastyprocedures wherein a balloon catheter is inserted into the arterialcirculation and then threaded (with radiographic contrast agents forvisualization) to a site of occlusion. In dilating the occluded artery,various forms of tissue damage and inflammatory reactions (e.g.,restenosis) can occur including ischemic tissue injury.

Specifically, toxic side effects of intra-arterial catheterization andinfusion of radiographic contrast agents prolong hospital stays, add tothe cost of medical care, and can be fatal. The incidence ofradiographic-contrast-agent-induced acute renal failure, currentlyestimated to be as high as 50 percent among patients with diabetesmellitus and preexisting renal disease who receive contrast agents, islikely to remain high as the use of invasive intra-arterial proceduresto diagnose and treat complex disease continues to grow.

Radiographic contrast agents are used in medical imaging. Medicalimaging is the production of images of internal organs and tissues bythe application of nonsurgical techniques. Contrast agents are chemicalsused to enhance the image, and to increase contrast between the targetorgan and surrounding tissues. Prevention or mitigation of renal failureafter the administration of a radiographic contrast agent has beennotably difficult. Calcium-channel antagonists, adenosine antagonists,and dopamine have all been used without convincing evidence of benefit.

Tepel et al. proposed the oral administration of approximately 1200 mgof N-acetylcysteine per day, given orally in divided doses on the daybefore and on the day of the administration of the radiographic contrastagent. (Tepel et al., New England J. Med., Jul. 20, 2000). Oraladministration allegedly prevented the expected decline in renalfunction in all patients with moderate renal insufficiency, andtherefore high risk, who were undergoing computed tomography.

NAC has been used successfully to ameliorate the toxic effects of avariety of experimentally or clinically induced ischemia-reperfusionsyndromes of the heart, kidney, lung, and liver. In each of thesesyndromes, it is thought that the activity of NAC is related to itsaction as a free-radical scavenger, or as a reactive sulphydryl compoundthat increases the reducing capacity of the cell. The specific mechanismof NAC to prevent the nephrotoxic effects of contrast agents is notknown.

Therefore, there is a need in the art to find better ways to usethiol-based chemoprotectants, such as NAC and STS and to take advantageof their pharmacokinetic properties. There is also a need in the art tofind better, higher dose cytotoxic treatment regimens for head and neckas well as brain tumors that avoid dose-limiting due to side effects.

There is a need in the art for a compound that can be used withintra-arterial catheterization procedures to reduce organ toxicity.Diabetic patients with markedly reduced renal function, in whom coronaryangiography is often delayed because of the considerable risks to renalfunction entailed by angiography, may particularly be benefited bytargeted delivery of a protectant agent. Additionally, there is a needin the art for a low cost compound which is generally available, easy toadminister and has limited side effects. There is a need in the art fora compound and a method of administration of the compound that can beused to reduce or eliminate tissue damage caused by intra-arterialprocedures.

Additionally, there is a need in the art to find better ways to usethiol-based radiographic protectants, such as NAC and STS (sodiumthiosulfate) and to take advantage of their pharmacokinetic properties.There is also a need in the art to find an agent protective againstintra-arterial catheterization-induced reductions in organ function.These and other problems of the prior art are solved by the presentmethod and pharmaceutical composition.

SUMMARY OF THE INVENTION

The present invention provides a method and compound for locallyadministering a thiol based chemoprotectant to treat or mitigate theside effects of cytotoxic cancer therapy for brain tumors located in thehead or neck. Further, the present invention provides a method andcompound for locally administering a thiol-based chemoprotectant to anorgan or tissue to protect against injury from diagnostic or therapeuticintra-arterial procedures. The method includes administering athiol-based chemoprotectant agent intra-arterially in conjunction with,before, or after administration of a cytotoxic agent.

In one embodiment, the cytotoxic agent is a cancer chemotherapeuticagent. The cytotoxic agent is dose-limited due to myelosuppressiveeffects systemically. The cancer chemotherapeutic agent is selected fromthe group consisting of cis-platinum compounds, taxanes (e.g.,paclitaxel), steroid derivatives, anti-metabolites, vinca alkaloids,adriamycin and doxorubicin, etoposide, arsenic derivatives,intercalating agents, alkylating agents (such as melphalan) andcombinations thereof. The cytotoxic agent is administered within eighthours (before, during or after) of the thiol-based chemoprotectant agentadministration. In yet another embodiment, when the tumor is located inthe head or neck or brain, the cytotoxic agent is administered such thatthe majority of the dose is directed to the head or neck region.

Preferably, the thiol-based chemoprotectant agent is a compound selectedfrom the group consisting of N-acetyl cysteine (NAC), sodium thiosulfate(STS), GSH ethyl ester, D-methionine, Ethyol, and combinations thereof.In one embodiment, the thiol-based chemoprotectant agent is administeredin a pyrogen-free sterile solution by a catheterization procedure via acatheter having a tip that is located in the descending aorta. In yetanother embodiment, the dose of the thiol-based chemoprotectant agentper procedure is from about 200 mg/m² to about 40 g/m². Most preferably,the dose of NAC agent per procedure is from about 400 mg/m² to about1200 mg/m² and the dose of STS is from about 5 g/m² to about 40 g/m².

In one embodiment, the intra-arterial catheter is positioned in anartery providing blood flow to a potential site or organ of injury. Inyet another embodiment, the intra-arterial administration is at a sitein the descending aorta. In yet another embodiment, the injury is causedby injecting a cytotoxic agent including a radiographic contrast agentusing an intra-arterial catheter.

In yet another embodiment, the thiol-based chemoprotectant agent isadministered in a pyrogen-free, non-oxidized sterile solution having areducing agent, a buffer to maintain pH at or near physiologic pH and ametal chelating agent to bind metal ions that can catalyze oxidation ofthe thiol-based chemoprotectant agent. Preferably, the reducing agent isselected from the group consisting of vitamin E, tocopherol,dithiotreitol, mercaptoethanol, glutathione, and combinations thereof.Preferably, the buffer is one that is relatively non-toxic and canmaintain a pH of between 6 and 8 (e.g., phosphate buffer, Tris buffer).Preferably, the thiol-based chemoprotectant agent is stored in a vialhaving a blanket of an inert gas. Most preferably, the inert gas isselected from the group consisting of argon, helium, nitrogen andmixtures thereof.

The present invention further provides a pharmaceutical composition fortreatment to protect against injury from diagnostic or therapeuticintra-arterial procedures, and for head and neck brain tumors. Thecompound includes a first agent for intra-arterial administration and asecond agent administered intra-arterially.

Preferably the second agent is a thiol-based chemoprotectant agent. Inone embodiment, the second agent is administered to the descending aortaor further downstream.

The first agent is administered to a carotid or vertebral artery. Thefirst agent is a radiographic contrast agent delivered intra-arteriallyto position a catheter. In one embodiment, the first agent isadministered within eight hours (before, during or after) of the secondagent. The second agent is administered immediately after intra-arterialcatheterization, prior to radiographic contrast agent, to within eighthours of the radiographic contrast agent.

In one embodiment, the second agent is administered in a pyrogen-free,sterile solution. In further embodiments, the solution is non-oxidizedand has a reducing agent, a buffer to maintain pH at or near physiologicpH and a metal chelating agent to bind up metal ions that can catalyzeoxidation of the thiol-based chemoprotectant agent. Preferably, thereducing agent is selected from the group consisting of vitamin E,tocopherol, dithiothreitol, mercaptoethanol, glutathione, andcombinations thereof. Preferably, the buffer is one that is relativelynon-toxic and can maintain a pH of between 6 and 8 (e.g., phosphatebuffer, Tris buffer).

In one embodiment, the thiol-based chemoprotectant agent is stored in avial having a blanket of an inert gas. Most preferably, the inert gas isselected from the group consisting of argon, helium, nitrogen andmixtures thereof. Preferably, the thiol-based chemoprotectant agent is acompound selected from the group consisting of N-acetyl cysteine (NAC),sodium thiosulfate (STS), GSH ethyl ester, D-methionine, Ethyol, andcombinations thereof. Preferably, the dose of the thiol-basedchemoprotectant agent per procedure is in the range of 200 mg/m² to 2000mg/m². In a further preferred embodiment, the dose of NAC per procedureis in the range of 400 mg/m² to 1200 mg/m².

Further, one advantage of NAC is that it is protective against multipleintra-arterial procedure toxicities, including but not limited to thosecaused by radiographic contrast agents.

The methods and compounds will best be understood by reference to thefollowing detailed description of the preferred embodiment, taken inconjunction with the accompanying drawings. The discussion below isdescriptive, illustrative and exemplary and is not to be taken aslimiting the scope defined by any appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an anatomical diagram of major arteries and the top levelfor placing the catheter for administration of the thiol-basedchemoprotectant agent.

FIG. 2 (A-C) shows the effect of bone marrow recovery and lower nadirsusing the left carotid aortic infusion inventive method withchemotherapy and the chemoprotectant NAC. Specifically, FIG. 2A showsthe effect on white blood cells, FIG. 2B the effect on platelets andFIG. 2C the effect on granulocytes.

FIG. 3A shows dose/response for N-acetylcysteine chemoprotection. FIG.3B shows dose/response for D-methionine chemoprotection. Cytotoxicitywas assessed in cultured LX-1 SCLC cells, 1×10⁴ cells per well in 96well plates, using the WST colorometric assay. Cells were treated withapproximately 90% lethal dose of chemotherapy (melphalan=20 μg/ml,carboplatin=200 μg/ml, cisplatin=20 μg/ml). Chemoprotectant was added atthe indicated concentration of N-acetylcysteine shown in FIG. 3A orD-methionine shown in FIG. 3B either alone (n) or immediately followingchemotherapy. Data are expressed as the percentage of live cellscompared to untreated control samples (without chemotherapy) and eachpoint represents the mean±standard deviation of 4 wells.

FIGS. 4A and 4B show cytoenhancement and chemoprotection. FIG. 4A showsthe effect of BSO and N-acetylcysteine on Carboplatin cytotoxicity. FIG.4B shows the effect of BSO and N-acetylcysteine on etoposide phosphatecytotoxicity. Cytotoxicity was assessed in cultured LX-1 SCLC cells,1×10⁴ cells per well in 96 well plates, using the WST colorometricassay. BSO cytoenhancement consisted of preincubation at 100 μM BSO for18 hours. The chemoprotective agent was added immediately afterchemotherapy. The experimental conditions were dose/responses forchemotherapy (carboplatin or etoposide phosphate) alone (m), BSOcytoenhancement (

), N-acetylcysteine rescue, (1000 μg/ml N-acetylcysteine, n), or BSOcytoenhancement and N-acetylcysteine rescue (u). Data are expressed asthe percentage of live cells compared to untreated control sampleswithout chemotherapy and each point represents the mean±standarddeviation of 4 wells.

FIG. 5 shows cytoenhancement and chemoprotection in fibroblasts.Cytotoxicity was assessed in GM294 human fibroblasts, 1×10⁴ cells perwell in 96 well plates, using the WST colorometric assay. Cells werepretreated with or without BSO, 100 μM for 18 hours prior to addition ofchemotherapeutics (melphalan=10 μg/ml, carboplatin=100 μg/ml,cisplatin=7.5 μg/ml, etoposide phosphate 100 μg/ml) either alone (openbar), or with N-acetylcysteine rescue, (1000 μg/ml N-acetylcysteine,striped bar), BSO cytoenhancement (black bar), or BSO cytoenhancementand N-acetylcysteine rescue (cross hatched bar). Data are expressed asthe percentage of live cells compared to control samples (withoutchemotherapy) and each point represents the mean±s.d. of 4 wells.

FIG. 6 shows time dependence for rescue of chemotherapy cytotoxicity.Chemotherapy cytotoxicity was assessed in cultured LX-1 SCLC cells(1×10⁴ cells/well in 96 well plates) using the WST colorometric assay.Cells were treated with melphalan at 20 μg/ml, carboplatin 200 μg/ml,cisplatin 10 μg/ml, or etoposide phosphate 200 μg/ml. Cells thenreceived either no protectant (open bars), or sodium thiosulfate, 2000μg/ml, added immediately (striped bars), 2 hours (black bars) or 4 hours(cross hatched bars) after chemotherapy. Data are expressed as thepercentage of live cells compared to control samples (withoutchemotherapy) and each point represents the mean±standard deviation of 4wells.

FIGS. 7A and 7B show the effect of cytoenhancement and chemoprotectionon apoptosis. FIG. 7A shows caspase-2 enzymatic activity. FIG. 7B showsTUNEL staining for DNA fragmentation. Apoptosis was assessed in culturedLX-1 SCLC cells pretreated for 18 hours with or without 100 μM BSO. Theexperimental conditions were no addition (open bars), melphalan (10μg/ml, striped bars) or melphalan (10 μg/ml) plus N-acetylcysteine (1000μg/ml) (cross hatched bars) for 20 h prior to harvest. Caspase-2activity is expressed as percentage activity in untreated controlsamples (mean±standard deviation, n=3). TUNEL staining is expressed asthe percentage of cells showing positive staining.

FIG. 8 shows the results of NAC delivery to rat brain. Radiolabeled NACin combination with unlabeled low dose NAC (140 mg/kg) or high dose NAC(1200 mg/kg) was administered to rats with the following routes ofinfusion: i.v. (open bars), intra-arterially into the right carotidartery (striped bars), intra-arterial via the left carotid artery withleft internal artery occlusion (aortic infusion) (black bars), andintra-arterial (right carotid) with BBBD (cross hatched bars).Radiolabel in tissue homogenates is expressed as the mean and standarderror of the % administered dose of 14C-NAC per gram of tissue (n=3 ratsper group). Significant differences from i.v. delivery are indicated by*P<0.05 and **P<0.001.

FIG. 9 represents tests showing the effect of NAC route ofadministration on mortality +/−BSO. A Kaplan-Meier product limitanalysis was used to evaluate the mortality due to chemoprotection withNAC. Rats were treated with or without BSO (10 mg/kg i.p. b.i.d.×3,black bars) prior to treatment with chemotherapy (Carboplatin 200 mg/m2,etoposide phosphate 100 mg/m2, melphalan 10 mg/m2). Chemoprotectionconsisted of NAC (1200 mg/m2) or NAC (1000 mg/m2) plus STS (8 g/m2)given either i.v. (n=17 BBSO, n=8+BSO) or by aortic infusion (n=19 BBSO,n=28+BSO). P=0.0014 by Wilcoxon analysis.

FIGS. 10A-10D show the results of tests on chemoprotection forchemotherapy-induced myelosuppression. Rats received tri-drugchemotherapy (Carboplatin 200 mg/m2, etoposide phosphate 100 mg/m2,melphalan 10 mg/m2), with (triangles, squares) or without (circles)chemoprotection. Blood counts were determined prior to chemotherapy, atthe blood nadir (6 days), and in the recovery phase (9 days aftertreatment). Chemoprotection was with NAC (1200 mg/kg) administered viaaortic infusion 30 min prior to chemotherapy (triangles) or NAC prior tochemotherapy and STS (8 g/m2) immediately after chemotherapy (squares).In FIG. 10B and FIG. 10D animals received BSO (10 mg/kg i.p.×3 d) priorto treatment with chemotherapy. Panel A shows granulocyte counts withoutBSO FIG. 10B shows granulocyte counts with BSO (mean+/−SEM, n=6 pergroup) FIG. 10C shows platlet count without BSO

FIG. 10D shows platlet count with BSO. Significant difference from theno protectant groups were determined by Wilcoxon/Kruskal-Walliis ranksums tests (*p<0.05, **p<0.01).

DETAILED DESCRIPTION OF THE INVENTION

The present method includes administration of at least two differentagents for treating brain or head and neck tumors. The first agent is acytotoxic agent. In one example, the cytotoxic agent is radiation or achemotherapeutic agent. The radiation or chemotherapeutic agentgenerally has a dose-limiting systemic side effect of myelosuppression.If myelosuppression is too severe, it is life threatening as the patientis unable to generate enough white blood cells of multiple lineages tocoordinate immune surveillance function for defending against pathogenattack. Therefore, any ability to reduce bone marrow toxicity ormyelosuppression will allow for greater and more effectiveadministration of the cytotoxic agent.

Current treatment to reduce bone marrow side effects include recombinantgrowth factors that are lineage-specific. Such growth factors haveincluded EPO (erythropoietin) for red cells and G-CSF (granulocytecolony stimulating factor) or GM-CSF (granulocyte macrophage colonystimulating factor) for various lineages of infection-fighting whitecells. In addition, TPO (thrombopoietin) is in clinical trials foraugmenting a platelet response in myelosuppressed patients. However,such growth factors act to stimulate lineage specific precursor cells todivide and mature down lineage-specific paths. Thus, the use of growthfactors results in a more rapid recovery from bone marrow toxicity butdoes not generally reduce the nadir of toxicity. Such growth factorshave been able to allow a patient to tolerate a greater number ofcytotoxic treatments (where myelosuppression is a limiting toxicity),but generally not higher doses of the cytotoxic agent administered.

The method allows for greater doses of the cytotoxic agent directed tohead and neck tumors. Specifically, the cytotoxic agent (if it is achemical compound or combination of compounds) is administeredintra-arterially such that it is directed initially to the head and neckcirculation. Thus, the highest concentration of cytotoxic agent isdirect to the location of the tumor to be treated. By contrast, ivadministration provides for the same concentration of cytotoxic agent inthe bone marrow, where side effects happen, as systemic dose.

Thiol-based chemoprotectant agents are nonspecific chemoprotectantagents. They are not specific to “normal” tissue or cells but canprotect both normal and tumor tissue. Earlier attempts to utilize thechemoprotectant properties of thiol-based chemoprotectant agents havefailed due to the fact that they were administered either orally orsystemically, they were rapidly metabolized, and protected both normaland tumor tissue. Systemic administration includes iv.

The method includes utilizing a spatial two-compartment pharmacokineticmodel which results in a general tissue first pass effect to preventsignificant or chemoprotectant doses of thiol-based chemoprotectantagents from gaining general systemic circulation through the venouscirculatory system. The method utilizes only one pass going to tissuesbelow the level of the heart in order to effect a chemoprotectanteffect. Therefore, head and neck tumors are treatable throughregionalization of doses of the cytotoxic agent to the brain or head andneck where the tumor tissue is located and doses of the chemoprotectantto general tissues below the level of the heart where the majority ofbone marrow tissue is located.

The ability of a thiol-based chemoprotectant agent to show a first passeffect through non-liver tissue was surprising. Once any thiol-basedchemoprotectant agent that is not tissue-absorbed gains access to thevenous circulation, it will be cleared through a liver first pass orrapidly removed through renal clearance. When administered not accordingto the method described, NAC is actively transported across the BBB. Anexample of spatial compartmentalization is the administration of athiol-based chemoprotectant agent into the descending aorta or lowerpreventing any significant chemoprotectant concentrations of thethiol-based chemoprotectant agent from ever reaching the brain or heador neck region where the tumor tissue is located. Spatialcompartmentalization is facilitated by the rapid tissue uptake of thethiol-based chemoprotectant agent. Spatial compartmentalization isneeded in order to achieve any meaningful therapeutic benefit with athiol-based chemoprotectant agent. Without spatial compartmentalization,the undesirable result of the thiol-based chemoprotectant agentprotecting the tumor tissue will occur. In the case of NAC as thethiol-based chemoprotectant agent, passage of the BBB by NAC willrestore glutathione levels to the brain tumor, thereby increasing thetumor's resistance to cytotoxic drugs.

The ability to set up a two-compartment pharmacokinetic model wasdiscovered through a series of experiments using in vitro and in vivomodels and administering both chemotherapeutic agents and thethiol-based chemoprotectant agents NAC and STS. However, it should benoted that radiation therapy can be similarly localized or even moreeasily localized than chemotherapeutic agents. Tissue culturepharmacological studies have shown that treatment with NAC and STS canreduce cell killing by the chemotherapeutics melphalan, carboplatin, andcisplatin. The in vivo results show that localized NAC or STSadministration resulted in less myelosuppression and faster recoveryfrom chemotherapy. Moreover, synergistic results were observed with thecombination of thiol-based chemoprotectant agent NAC and STS providingevidence for a combination of thiol-based chemoprotectant agents toenhance the protective role when the combination of thiol-basedchemoprotectant agents is administered according to the inventiveprocess.

As shown in FIG. 1, in another embodiment of the method, a thiol-basedchemoprotectant agent is administered intra-arterially such that it isdirected systemically. This provides the highest concentration ofthiol-based chemoprotectant agent to the location of organ damage, forexample the kidneys, to protect against reduction of renal function.Oral administration, by contrast, provides for general systemicadministration with the concentration of protective agent goingelsewhere in the body, mainly the liver, and requiring higher dosages soas to provide a sufficient dose at the kidney (often the site of organdamage for radiographic contrast agents). In addition, the thiol-basedchemoprotectant agent is not specific to normal tissue or cells but canprotect both normal and tumor tissue.

The method is based upon results obtained utilizing a spatialtwo-compartment pharmacokinetic model that resulted in a general tissuefirst pass effect to prevent significant or radiographic-protectantdoses of thiol-based radiographic-protectant agents (specificallyillustrated are NAC and STS) from gaining general systemic circulationthrough the venous circulatory system. Thus, there was a surprising needfor only one pass going to tissues of the renal system. This resultprevented decreased renal function through regionalization of doses ofthe radiographic agent to the area where radiography is to be performedand doses of the protectant to the renal system.

As shown in FIG. 1, a catheter was inserted into the circulatory systemgenerally via the femoral artery. In the first embodiment, a catheter isinserted into the body and administers a dosage of radiographic contrastagent into the body. An effective dosage of the thiol-based protectiveagent, preferably NAC, is administered at any time from immediatelyafter the intra-arterial catheterization, preferably before contrastagent, to within about eight (8) hours after the administration of theradiographic contrast agent. The catheter for delivery of theradiographic protective agent is preferably inserted into the arterialsystem downstream of the aorta and directed in the mesenteric arterysystem. The thiol-based protective agent is introduced into the body atany time within about 5 hours after administration of the radiographiccontrast agent to reduce or eliminate renal failure or decrease in renalfunction associated with administration of a renal contrast agent.

In a further embodiment, a catheter is inserted into the circulatorysystem (e.g., femoral artery) and administers an effective dosage of athiol-based protective agent into the body. A radiographic contrastagent is administered by the same catheter and is used to position thecatheter to the appropriate place for the therapeutic or diagnosticprocedure. The thiol-based protective agent is introduced into the body,generally via the same arterial catheter, at any time within about 5hours before or after administration of the radiographic contrast agent.Preferably, the thiol-based protective is administered via the arterialcatheter one or a plurality of times during the procedure.

Pharmaceutical Formulations

Techniques for the formulation and administration of the compounds ofthe instant application may be found in “Remington's PharmaceuticalSciences” Mack Publishing Co., Easton, Pa., latest addition. Suitableroutes of administration are intra-arterial.

The compositions and compounds of the present invention may bemanufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, emulsifying, encapsulating, entrapping,or lyophilizing processes. Pharmaceutical compositions for use inaccordance with the present invention thus may be formulated inconventional manner using one or more physiologically acceptablecarriers comprising excipients and auxiliaries that facilitateprocessing of the active compounds into preparations, which can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

For injection, the compounds of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers,such as Hank's solution, Ringer's solution, or physiological salinebuffer. The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulary agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use. In one embodiment, a reducing agent or an anti-oxidant agentis added to the formulation of the thiol-based protective agent toprevent oxidation of the thiol-based protective agent. The antioxidantmay include, but is not limited to, vitamin E, tocopherol,dithiotreitol, mercaptoethanol, glutathione. In one embodiment, an inertor non-oxidizing gas is added to a vial for intra-arterialadministration. Examples of such gasses are nitrogen, argon, helium, andcombinations/mixtures thereof.

A therapeutically effective dose refers to that amount of the compoundthat results in a reduction in the development or severity ofmyelosuppression. In another embodiment, toxicity and therapeuticefficacy of such compounds can be determined by standard pharmaceutical,pharmacological, and toxicological procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratiobetween LD₅₀ and ED₅₀. Compounds that exhibit high therapeutic indicesare preferred. The data obtained from cell culture assays or animalstudies can be used in formulating a range of dosage for use in humans.The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. (See e.g. Finglet al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1).

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

According to the method, a thiol-based chemoprotectant agent isadministered intra-arterially in order for systemic tissues to beexposed to an initial dose of the thiol-based chemoprotectant agent inhigh enough concentration to provide a chemoprotective effect and tobypass the venous circulation and be eliminated by the liver. NAC isactively transported across the BBB. In one embodiment, to preventaccess to the brain, after a transfemoral carotid/vertebral arterycatheter is placed to perfuse the brain, the catheter can then besubsequently refracted and placed in the descending aorta for NACinfusion, thus performing a single surgical procedure with one catheter.This allows for minimal risk from arterial catheter procedures only andfor high concentrations of NAC to be delivered to peripheral tissues andorgans but not to brain and head.

Synthesis

Each thiol-based chemoprotectant agent, such as NAC or STS, can besynthesized by convention methods and are commercially available as asterile solution.

Example 1

This example shows the results of an in vivo experiment in rats having acatheter implanted in the descending aorta for NAC administration. Therats were set up for drug administration by pushing a catheter forwardpast the junction of the external and internal carotid arteries (towardthe aorta), and temporarily sealing off the internal carotid for goodmeasure so nothing goes to the brain (left carotid aortic infusionmethod). In patients where entry is via the femoral artery and thecatheter is threaded through the aorta to get to the brain in the firstplace, one would just pull the catheter back to the aorta to do the NACinfusion.

Rats were treated with carboplatin (200 mg/m²), melphalan (10 mg/m²) andetoposide phosphate (100 mg/m²). In the NAC animals, NAC was infusedwith the left carotid aortic infusion method immediately after thechemotherapeutic agents, at concentrations ranging from 400-1200 mg/m².White blood cells (wbc) and platelets (plt) were counted at baselinebefore the experiment and at 6 and 9-11 days after treatment with thechemotherapeutic agents. The animals that did not receive NAC weretested on day 9-10 while the NAC animals were tested on day 10-11.Counts are in 1000s per μl blood. These data in the initial experimentshown in Table 1 provide initial results without a white blood celleffect. The lack of white blood cell effect was not repeated as therewere significant effects shown below.

TABLE 1 chemo wbc wbc wbc plt plt plt alone base d 6 d 9/10 base d 6 d9/10 mean 11.4 0.8 3.2 778 63 164 +/−sd +/−4.1 +/−0.2 +/−1.6 +/−233+/−59 +/−91 number 8 4 3 7 4 3 chem + wbc wbc wbc plt plt plt NAC base d6 d 10/11 base d 6 d 10/11 mean 7.9 0.5 4.8 817 101 1232 ** +/−sd +/−2.1+/−0.2 +/−0.7 +/−142 +/−48 +/−167    number 5 4 3 5 4 3

The data in table 2 show additional results. In the NAC animals, NAC wasinfused by the left carotid aortic infusion method 30 min prior tochemo, at a concentration of 1200 mb/m2. White blood cells (wbc) andplatelets (plt) were counted at baseline before the experiment and at 6and 9 days after treatment with the chemotherapeutic agents. The data inTable 1 show that NAC treatment decreased the nadir blood count for bothwhite cells and platelets, and blood counts recovered from chemotherapyfaster.

TABLE 2 wbc wbc wbc plt plt plt base d 6 d 9 base d 6 d 9 chemo alonemean 6.4 1.6 5.2 878 187 599 +/−sd +/−0.3 +/−0.4 +/−0.7 +/−46 +/−70+/−187 number 6 6 6 6 6 6 chem + NAC mean 5.1 3.3 6.2 721 388 1155 +/−sd+/−0.4 +/−0.7 +/−1.6 +/−59 +/−95 +/−154 number 6 6 6 6 6 6

These data show that with NAC the platelets recovered from chemotherapyfaster.

In addition, the experiment with the same chemotherapy agents at thesame doses with and without BSO (buthionine sulfoximine) shows improvedwhite blood cell recovery and higher nadirs with the chemoprotectant(NAC alone or with STS at the doses listed above) in FIG. 2A. Similardata are shown with platelets in FIG. 2B, and with granulocytes in FIG.2C.

Example 2

This example shows the results of NAC delivery by different means ofcatheter-based administration. In this experiment, radiolabeled NAC wasadministered to rats by three different routes. Liver and kidney tissue,in addition to the ipsilateral and contralateral hemispheres of thebrain, were analyzed for radioactivity. Results are shown as the percentof the injected radioactive dose per gram of tissue. Route indicatesintravenous (i.v.), intra-arterial into the right carotid artery (i.a.),or left carotid with internal artery occlusion and aortic infusion (leftcarotid aortic infusion). The left carotid aortic infusionintra-arterial method uses descending aorta placement of the cathetertip and NAC administration.

route left hem right hem liver kidney number i.v. 0.02 0.03 0.59 0.72 2i.a. 0.03 0.41 0.57 0.70 3 Left carotid 0.04 0.04 0.29 1.42 2 aorticinfusion

These data show that i.a. delivery provided much brain delivery in theinfused hemisphere. When the NAC was administered i.v., negligibleamounts were found in brain (0.03% of the injected dose, n=2). Whenradiolabeled NAC was administered intraarterially into the right carotidartery of the rat, high levels of radiolabel were found throughout theright cerebral hemisphere. Delivery was 0.41% of the injected dose(n=3), comparable to the levels found in liver (0.57% of the injecteddose) or kidney (0.70% of the injected dose). In contrast, the leftcarotid aortic infusion method prevented brain delivery. The Leftcarotid aortic infusion method also changed bio-distribution inperipheral tissues in that liver delivery was decreased and kidneydelivery was increased. The change in tissue delivery with differentmodes of administration is likely due to NAC being related to the aminoacid cysteine that is rapidly bound by tissues via the amino acidtransporters. In summary, the method of administration of NAC markedlyaffected its biodistribution.

Example 3

This example tested whether the inventive method for intra-arterialinfusion (via the left carotid artery, with left internal arteryocclusion) could reduce brain delivery of NAC and increase systemicdelivery. Brain delivery was 0.04% of the injected dose with theinventive method (n=2).

In conjunction with other pharmacological and physiological data, theseresults show that NAC is protective when N-acetylcysteine isadministered prior to, (preferably 30 minutes prior to), the cytotoxicagent at a dose which provides a serum concentration of NAC of between0.5 mM to 15.0 mM, preferably 5 mM to 12.5 mM. Generally, a dose ofbetween 40.0 mg/kg to 1000 mg/Kg of N-acetylcysteine will provide anappropriate serum concentration in humans and other mammals.

Example 4

This example shows the inventive process being used to compare bonemarrow toxicity of a chemotherapeutic agent (alone or in combination) toadministration of NAC alone (1000-1200 mg/m² alone or in combination)and NAC plus STS. The chemotherapy agents were carboplatin (200 mg/m²)melphalan (10 mg/m²) and etoposide phosphate (100 mg/m²). The dose ofSTS in the rat was 8 g/m² that is the equivalent to 20 g/m² in humans.The method of administration was left carotid aortic infusion method.These data are expressed according to lineages of bone marrow cell inTable 3a and 3b.

TABLE 3a wbc wbc wbc Platelet Plt plt chemo mean 9.2 0.7 3.2 759.0 63.3164.0 sd 3.6 0.2 1.6 260.5 59.0 91.0 n 7 4 3 7 4 3 wbc wbc Wbc PlateletPlt plt chemo+ mean 7.1 1.4 5.2 772.3 132.0 1559.0 sd 2.7 1.1 1.7 171.536.8 567.1 n 4 2 2 4 2 2 wbc wbc wbc Platelet Plt plt chemo + mean 9.52.8 8.6 936.5 554.0 1950.0 NAC+ sd 6.0 0.4 3.6 186.0 147.1 134.4 n 2 2 22 2 2

TABLE 3b wbc wbc wbc plt plt plt base d 6 d 9 base d 6 d 9 chemo + BSOmean 6.8 0.8 5.4 930 92 387 +/−sd +?−0.8 +/−0.2 +/−1.1 +/−63 +/−28+/−139 number 11 11 6 11 11 6 BSO + chem + NAC mean 8.4 2.2 7.6 818 3411560 +/−sd +/−1.5 +/−0.3 +/−1.2 +/−99 +/−79 +/−194 number 9 9 6 9 9 6

These data show the synergistic effect of a combination of STS and NACas the combined thiol-based chemoprotectant agents administeredaccording to the inventive process.

Example 5

This example provides the results of an in vitro experiment using acombination of a taxane (paclitaxel) with a paclitaxel-cytotoxicenhancing agent BSO and a glutathione-reviving agent NAC in culturedtumor cells. Paclitaxel is cytostatic in cultured cells atconcentrations from 1 to 10 micromolar, that is, the tumor cells do notgrow but they are not killed either. At 20 uM, paclitaxel begins to becytotoxic and at 30 uM it is completely toxic in cultured tumor cells.When the tumor cells were pretreated cells with BSO, paclitaxel additionwas completely cytotoxic in vitro at doses as low as 5 uM.

NAC did not change the dose response for paclitaxel alone. However, NACcompletely reversed the enhanced toxicity from the BSO treatment,returning paclitaxel concentration effects to the non-BSO level. Thisexperiment was repeated twice with the data provided in Table 4.

TABLE 4 Treatment paclitaxel dose live cells (WST fluorescence)paclitaxel alone 5 1.274 +/− .071 20 0.771 +/− .056 paclitaxel + NAC 51.369 +/− .061 20 0.823 +/− .094 BSO + paclitaxel 5   0.056 +/− .004 **20   0.045 +/− .004 ** BSO + paclitaxel + NAC 5  1.419 +/− .095 * 20 0.732 +/− .100 * * significantly different from BSO and paclitaxel **significantly different from paclitaxel alone

Example 6 Chemoprotection Against Cytotoxicity

The dose/response for rescue from chemotherapy cytotoxicity wasevaluated for four different small molecular weight sulfur containingchemoprotectants. Each chemotherapeutic agent was used at aconcentration affording approximately 90% lethality in the absence ofBSO (20 μg/ml melphalan, 200 μg/m1 carboplatin, 15 μg/m1 cisplatin).Over all, N-acetylcysteine was the most effective of the thiol agentstested, on a μg/ml basis. The concentration dependence for protectionwith N-acetylcysteine in comparison to D-methionine is shown in FIGS. 3Aand 3B, and Table 5 shows the EC50 for protection afforded by eachprotective agent. As shown in FIG. 3A AND Table 5, the cytotoxicity ofeach alkylator was reduced by 75-90% by concurrent administration ofN-acetylcysteine, but N-acetylcysteine was more active against melphalan(EC50=74±18 μg/m1) than the platinum agents. In contrast, as shown inFIG. 3B and Table 5, D-methionine did not protect against melphalantoxicity at the doses tested (50 to 1000 μg/ml), although it was highlyprotective against cisplatin toxicity, with a half-maximal concentrationof 140±41 μg/ml. The maximum magnitude of protection was variablebetween experiments, ranging from about 70% to 100% protection, andprotection was consistently less for carboplatin than for cisplatin ormelphalan. All agents tested required a significantly higher dose toprotect against carboplatin than against cisplatin or melphalan. On aμg/ml basis, glutathione ethyl ester was the least effective protectiveagent.

Chemotherapy agents were fixed at the 90% lethal dose for each agent.Each EC50 measurement comprised 6 concentrations with 4 wells perconcentration, and each dose response was performed twice for eachprotectant. EC50 concentrations, in μg/ml are reported as theaverage±pooled standard deviation for two independent experiments. Forthe combination of D-methionine with melphalan, protection was notdetected. For each chemoprotectant, t test comparisons were done formelphalan versus cisplatin, cisplatin versus carboplatin, andcarboplatin versus melphalan, and significant differences are indicated(**=P<0.01; ***=P<0.001).

TABLE 5 Protection against chemotherapy cytotoxity Chemoprotective agentEC50 (μg/ml) Sodium Glutathione chemotherapy N-acetylcysteinethiosulfate D-methionine ethyl ester Melphalan  74 ± 18 *** 110 ± 78   None ***  303 ± 124 *** Cisplatin 151 ± 15 **  86 ± 76 ** 140 ± 41 *** 530 ± 30 *** Carboplatin 200 ± 15 *** 442 ± 203 ** 379 ± 123 *** 995 ±48 ***

Example 7 Cytoenhancement and Chemoprotection in Combination

The effects of BSO cytoenhancement and thiol chemoprotection on thedose/response relationships for cytotoxicity of the alkylatingchemotherapeutics were evaluated in the B.5 LX-1 cells. BSOcytoenhancement consisted of preincubation with 100 μM BSO for about18-24 hours prior to addition of chemotherapy, and rescue consisted of1000-2000 μg/ml of thiol chemoprotectant added immediately afterchemotherapy. As shown in FIG. 4A and Table 6, BSO consistentlydecreased the EC50 for cytotoxicity and increased the maximum degree oftoxicity. The specific case of carboplatin and N-acetylcysteine is shownin FIG. 4A. Glutathione depletion with BSO increased carboplatincytotoxicity, reducing the EC50 by 48% (P<0.01). As detailed in Table 6,similar BSO cytoenhancement was found with melphalan (53% reduction ofEC50, P<0.001), while the EC50 for cisplatin was reduced only 29%(P<0.05). Chemoprotection with N-acetylcysteine blocked carboplatintoxicity as well as BSO-enhanced cytotoxicity. Similar chemoprotectionwas found with additional thiol agents, sodium thiosulfate andglutathione-ethyl ester, but D-methionine was only effective against theplatinum agents.

Cytoenhancement and chemoprotection against non-alkylatingchemotherapeutic agents was also evaluated. As shown in FIG. 4B,Glutathione depletion with BSO did not increase the cytotoxicity ofetoposide phosphate, nor did N-acetylcysteine decrease the cytotoxicityof etoposide phosphate. Similarly, no enhancement or protection wasfound with methotrexate or doxorubicin in the B.5 LX-1 cells, althoughcarcinoma cells of gastric origin showed some enhancement with BSO (datanot shown). Interestingly, although the growth inhibitory dose of taxol(approximately 10 nM) was not altered by BSO, glutathione depletion didshift the cytotoxic dose of taxol from 15 μM to 2 μM, and this enhancedcytotoxicity was completely reversed with N-acetylcysteine (not shown).

Whether the cytoenhancement and chemoprotection seen in the B.5 LX-1SCLC cells was a generalized phenomenon was evaluated by testing similarexperimental conditions in the GM294 human fibroblast cell strain. Cellswere treated with or without chemotherapy at the approximately halfmaximal dose found in the B.5 LX-1 cells, with or without pretreatmentwith BSO. As shown in FIGS. 3A and 3B, although melphalan, cisplatin andcarboplatin were all somewhat more cytotoxic in the fibroblasts ascompared to the tumor cells, nevertheless BSO enhanced the toxicity ofall three alkylators. In fibroblasts, N-acetylcysteine was partially tocompletely chemoprotective against the cytotoxicity induced bymelphalan, cisplatin and carboplatin, independent of BSO treatment. Asshown in FIG. 5, neither BSO cytoenhancement nor N-acetylcysteinechemoprotection affected the cytotoxicity of etoposide phosphate infibroblasts.

Half-maximal cytotoxic concentrations (EC50) are expressed as μg/ml.Each EC50 measurement comprised 6 concentrations with 4 wells perconcentration, and each dose response was performed in triplicate foreach chemotherapeutic agent. Data are reported as the mean±pooledstandard deviation for three independent experiments. P values are shownfor the reduction in EC50 by BSO treatment (*=P<0.05, **=P<0.01,***=P<0.001).

TABLE 6 Effect of BSO on the cytotoxicity of alkylatingchemotherapeutics Chemotherapy EC50 (μg/ml) Melphalan CisplatinCarboplatin Without BSO 13.8 ± 1.8    8.9 ± 2.4  103 ± 21    +BSO 6.4 ±0.6 *** 6.4 ± 0.9 * 55 ± 15 **

Example 8 Time Dependence for Chemoprotectant Rescue from ChemotherapyCytotoxicity

How long the addition of chemoprotectant could be delayed aftertreatment with chemotherapy and remain effective was evaluated. Cellswere treated with doses of chemotherapy providing approximately 90%lethality, for melphalan (20 μg/ml), carboplatin (200 μg/ml), orcisplatin (15 μg/ml). The thiol chemoprotectants were added eitherconcurrently with chemotherapy or up to 8 hours after chemotherapy. Formelphalan, chemoprotection was reduced if administration of STS wasdelayed for 2 hours, whereas sodium thiosulfate was still protective forthe platinum chemotherapeutics if delayed up to 4 hours after treatmentas shown in FIG. 6. Similarly, delayed administration ofN-acetylcysteine and glutathione ethyl ester reduced their protectiveactivity against melphalan cytotoxicity, whereas both agents maintainedprotective activity against platinum cytotoxicity. Separately, all threeagents were completely protective if added within 1 hour of melphalan,rather than 2 hours as shown in FIG. 6. Chemoprotection was noteffective against etoposide phosphate cytotoxicity at any time point.

The time dependence of D-methionine rescue of cisplatin cytotoxicity wasalso evaluated. Unlike chemoprotection with thiosulfate,N-acetylcysteine, or glutathione ethyl ester, the protection afforded byD-methionine was significantly reduced by delayed administration. Ifdelayed for 2 hours after cisplatin, D-methionine protection was reducedby 41.2±10.2% compared to the maximal protection seen with simultaneousaddition, while delaying D-methionine to 4 hours reduced protection by66.1±4.5% compared with simultaneous addition. Pretreatment withD-methionine for 30 min prior to addition of cisplatin did not increasethe amount of protection compared to simultaneous addition.

Example 9 Effects of Cytoenhancement and Chemoprotection on Apoptosis

Apoptosis was evaluated by measuring Caspase-2 enzymatic activity and byin situ TUNEL staining Treatment of B.5 LX-1 cells with melphalanresulted in an increase in Caspase-2 activity that was amplified by BSOpretreatment at low melphalan concentrations as shown in FIG. 7A. Theincrease in caspase activity was variable between experiments and rangedfrom 50-100% at 7-8 h to 250-600% at 20-24 h after treatment withmelphalan. TUNEL staining also demonstrated melphalan-induced apoptosis.In the experiment shown in FIG. 7B, TUNEL staining after melphalantreatment was positive in 29 of 3643 cells, compared to 7 of 4395 cellsin the untreated control, and BSO treatment prior to melphalan increasedthe positive staining to 800 of 1699 cells. In both the caspase-2 assayas shown in FIG. 7A and the TUNEL staining assay as shown in FIG. 7B,the effect of melphalan on apoptosis was reduced by the chemoprotectantN-acetylcysteine. In both assays, activity was maximal with low doses ofmelphalan, or with a 1 hour pulse treatment with the doses used in thecytotoxicity assays. Continuous treatment with the cytotoxic dose ofmelphalan actually reduced caspase-2 activity and TUNEL staining.

Cisplatin and carboplatin were less effective than melphalan at inducingcaspase activity. Over a range of doses (100, 150, or 200 μg/mlcarboplatin, and 5, 10, or 15 μg/ml cisplatin) and times (8, 12, 16, 20,24 h), each platinum agent increased Caspase-2 activity by 50-100%. Nosignificant amplification of caspase activity was induced by BSOtreatment. Additionally, no reduction in caspase enzymatic activitycould be detected after addition of N-acetylcysteine, and in someexperiments treatment with N-acetylcysteine actually increasedcisplatin- or carboplatin-induced caspase activity. Samples of the cellsused in the caspase and TUNEL assays were also evaluated for membranepermeability by trypan blue exclusion. In experiments producing negativeresults with the caspase-2 or TUNEL assays, trypan blue exclusion showedhigh numbers of non-viable cells after treatment with carboplatin orcisplatin and this was increased by BSO treatment.

Example 10 Biodistribution of Radiolabeled NAC

The method of administration of NAC and its biodistribution was tested.Intra-arterial infusion retrograde via the left external carotid artery,with transient left internal artery occlusion, was evaluated as amechanism to essentially perfuse via the descending aorta with limiteddelivery to the brain. When NAC was administered i.v., negligibleamounts were found in brain as determined by the percent administereddose of 14C-NAC per gram of tissue as shown in FIG. 8. Intra-arterialdelivery in the right internal carotid artery resulted in high levels ofradiolabel in the right cerebral hemisphere, and this was not increasedby BBBD. However, aortic infusion minimized brain delivery of NAC asshown in FIG. 8. At the low dose of NAC (140 mg/kg), aortic infusiondecreased liver delivery and increased kidney delivery, where as therewas no change in liver and kidney delivery at high dose (1200 mg/kg) NAC(data not shown). The serum concentration of NAC 10 min afteradministration of 1200 mg/kg was 2.4 0.6 mg/ml (n=6) as determined byradiolabel remaining in the blood.

Example 11 Toxicity of NAC

A dose escalation of NAC was performed in the rat. Initial doses of140-800 mg/kg NAC administered i.v. immediately after chemotherapy(n=4), were well tolerated but provided no detectable bone marrowprotection. Doses of NAC above 1200 mg/kg (n=3) were toxic whereas therewas no toxicity at 1200 mg/kg. Therefore, 1200 mg/kg of NAC was used forthe bone marrow protection studies, except when administered inconjunction with STS where a dose of 1000 mg/kg was used due to volumeconsiderations.

The toxicity of NAC was determined when infused for bone marrowprotection. As shown in FIG. 9. the mortality due to NAC wassignificantly dependent on the route of administration, with i.v.administration significantly more toxic than aortic infusion (P=0.0014).Pretreatment with BSO markedly enhanced the toxicity of NAC. Groupsgiven i.v. NAC after BSO treatment were halted early due to excessivemortality, with a stopping rule of 75% mortality within n=4 animals. Inselected animals (n=21) from all groups, blood pressure monitoringindicated most animals that expired experienced persistent acutehypotension, suggesting cardiac toxicity. Of particular note, however,there was no mortality nor any evidence of toxicity in 12 animals,without BSO, giving 1200 mg/kg NAC by aortic infusion 30 minutes priorto chemotherapy.

Example 12 Effect of NAC on Chemotherapy-Induced Bone Marrow Toxicity

Chemoprotection against chemotherapy-induced bone marrow toxicity wasdetermined in BSO treated and untreated animals given i.a. carboplatin,etoposide phosphate, and melphalan. NAC with or without STS wasadministered either about 30 minutes prior to chemotherapy orimmediately after chemotherapy, and was administered either i.v. or byaortic infusion. Chemoprotection was found with NAC as shown byincreased blood counts (white blood cells, granulocytes, platelets) atthe nadir, compared to no chemoprotectant as shown in Table 7 and Table8. Chemoprotection was effective whether or not animals were pretreatedwith BSO as shown in FIG. 10, and the magnitude of thechemotherapy-induced bone marrow toxicity nadir was minimized andrecovery to normal platelet levels was improved.

TABLE 7 Protection against chemotherapy-induced myelosuppression WhiteCell Granulocyte Platelet Treatment Rats Nadir Nadir Nadir No protectantN = 6 24.5 ± 5.8 20.7 ± 11.0 22.7 ± 8.7  NAC i.v. N = 6  46.0 ± 6.5*57.9 ± 8.8* 36.8 ± 10.4 post chemo NAC i.v. N = 8 51.4 ± 9.4 93.5 ± 28.153.3 ± 12.7 30 min prior NAC aortic N = 6 25.9 ± 4.9 36.6 ± 17.0 26.8 ±9.3  infusion post chemo NAC aortic N = 6  69.7 ± 19.3  206.2 ± 125.1*59.3 ± 17.9 infusion 30 min prior NAC aortic N = 6  53.8 ± 12.8*  83.4 ±21.3* 47.0 ± 11.5 infusion 30 min prior + STS post chemo The mean andstandard error are shown for nadir blood counts as a percent ofbaseline. *indicates P < 0.05 compared to no protectant, byWilcoxon/Kruskal-Wallis rank sums tests.

TABLE 8 Protection against BSO-enhanced chemotherapy- inducedmyelosuppression White Cell Granulocyte Platelet Treatment Rats NadirNadir Nadir No protectant  N = 11 12.9 ± 3.5 3.5 ± 1.3 9.1 ± 2.3 NACaortic N = 7 13.9 ± 4.2 19.8 ± 14.7 11.7 ± 3.5  infusion post chemo NACaortic N = 9 20.1 ± 2.7 115.4 ± 68.8* 23.1 ± 6.2  infusion 30 min priorNAC aortic N = 7  30.5 ± 6.5* 121.0 ± 40.2* 39.0 ± 7.4* infusion 30 minprior + STS post chemo The mean and standard error are shown for nadirblood counts as a percent of baseline. *indicates P < 0.05 compared tono protectant, by Wilcoxon/Kruskal-Wallis rank sums tests.

In animals that did not undergo BSO treatment, pretreatment with NAC(1200 mg/kg) by aortic infusion, with or without follow-up with STS, wasthe best treatment strategy as shown in FIGS. 10A and 10C. As shown inFIG. 10A, the chemotherapy-induced decrease in granulocyte counts wascompletely blocked (p<0.05) and platelet toxicity was reduced as shownin FIG. 10C by aortic infusion of NAC 30 minutes prior to chemotherapy.

In animals pretreated with BSO, good protection for granulocytes wasprovided by aortic infusion of NAC about 30 min prior to chemotherapy,but the best protection and the least mortality was provided by NACaortic infusion before and STS immediately after chemotherapy as shownin FIGS. 10B and 10D. As shown in FIG. 10B, the combinationchemoprotection (NAC and STS) regimen significantly blocked the toxicityfor granulocytes (p<0.01) and, as shown in FIG. 10D, platelets (p<0.01)compared to animals that received no chemoprotection. Chemoprotectionalso significantly enhanced the platelet recovery from chemotherapy. STSalone did not give consistent bone marrow protection (data not shown).

As noted, the discussion above is descriptive, illustrative andexemplary and is not to be taken as limiting the scope defined by anyappended claims.

All of the U.S. patents and patent applications listed in theApplication Data Sheet are incorporated by reference.

1. A method for treating or mitigating the side effects of cytotoxiccancer therapy comprising administering sodium thiosulfate (STS) inconjunction with a cis-platinum compound, wherein the STS isadministered about 4 hours after administration of the cis-platinumcompound, and wherein the STS is at a dose from 5 g/m² to 40 g/m². 2.The method for treating or mitigating the side effects of cytotoxiccancer therapy of claim 1 wherein the STS is administered intravenously.3. The method for treating or mitigating the side effects of cytotoxiccancer therapy of claim 1 wherein the STS is administeredintra-arterially.
 4. The method for treating or mitigating the sideeffects of cytotoxic cancer therapy of claim 3 wherein the cancer is acancer located in the brain, head or neck.
 5. A method for treating ormitigating the side effects of cytotoxic cancer therapy comprisingadministering sodium thiosulfate (STS) in conjunction with acis-platinum compound, wherein the STS is administered from about 4hours to 8 hours after administration of the cis-platinum compound, andwherein the STS is at a dose from 5 g/m² to 40 g/m².
 6. A method fortreating or mitigating the side effects of cytotoxic cancer therapycomprising administering sodium thiosulfate (STS) in conjunction with acis-platinum compound, wherein the STS is administered about 4 hoursafter administration of the cis-platinum compound, and wherein the STSis administered intravenously at a dose from 5 g/m² to 40 g/m².
 7. Themethod of claim 1 wherein administration of the cis-platinum compound isdirected to a region where the tumor is located and administration ofthe STS is directed to normal tissues.
 8. The method of claim 5 whereinadministration of the cis-platinum compound is directed to a regionwhere the tumor is located and administration of the STS is directed tonormal tissues.
 9. The method of claim 6 wherein administration of thecis-platinum compound is directed to a region where the tumor islocated.